Neuropeptides are a diverse assembly of small peptides that serve as neurotransmitters and neuromodulators in the central nervous system and as neurohormones in the periphery. The levels of these signaling molecules are known to be modulated by many centrally active agents and affected in a number of disease states-including psychotic disorders, Alzheimer's disease, and hypertension. Neuropeptides are inactivated by a group of proteolytic enzymes known as neuropeptidases. Therapeutic modulation of these enzymes can form the basis of effective treatments by altering the levels of their neuropeptide substrates, and they are therefore important drug targets. These efforts are hampered, however, both because substrate specificity is not understood and because there is a little structural information on the binding of substrates and inhibitors, which could be used for improving therapeutics. The goal of the proposed study is an understanding of specificity and function in neuropeptidases using endopeptidase 24.16 (neurolysin) as a model system. This enzyme, a zinc metallopeptidase, inactivates the neuropeptide neurotensin and may also participate in the inactivation of other neuroactive peptides. Endopeptidase 24.16 has two unusual properties shared by a number of neuropeptidases: it cleaves only short oligopeptides, and it recognizes a highly diverse set of cleavage sites. The basis for these unusual properties will be explored by an approach combining high- resolution structure determination and functional studies of the enzyme. We have determined the crystal structure of endopeptidase 24.16, which shows a deep active channel that prevents access by proteins. We will test the hypotheses that the length of substrates is further limited by the nature of the active site channel and that diverse sequences are accommodated by a high degree of plasticity in the catalytic region. Four specific aims are proposed: 1) to determine crystal structures with a series of peptides and inhibitors that will reveal the details of substrate binding, 2) to characterize the functional aspects of specificity through systematic variation of substrate features, 3) to identify important features of recognition by characterizing a closely related endopeptidase, 4) to test and refine our models of recognition by modulating and altering the specificity of the enzyme through mutagenesis. This work will define the general mechanisms of neuropeptide inactivation and provide a detailed view of inhibitor binding, laying the foundation for future therapy development.